ABSTRACT Youth with prediabetes (impaired glucose tolerance) or type 2 diabetes (T2D) experience relatively rapid beta-cell failure that is not prevented or delayed by metformin or insulin. Mechanisms underlying the fulminant course of T2D and the lack of preventive benefit from metformin and insulin in youth are unknown. Epigenetic biomarkers such as microRNAs (miRNAs) regulate genes important in glucose and insulin metabolism and may play a role in mediating responsiveness to metformin or insulin. miRNAs are short non-coding RNAs that regulate expression of genes important to glucose homeostasis, but their role in systemic responses to metformin and insulin is poorly understood, especially in youth. Our objective is to identify circulating miRNAs related to insulin resistance and beta-cell failure despite pharmacotherapy in youth. To achieve this goal, we propose a secondary analysis of the Restoring Insulin Secretion (RISE) Pediatric Mediation Study, a randomized controlled trial of glargine insulin followed by metformin or metformin alone in youth ages 10?19 with dysglycemia. We will assay circulating miRNAs using RNA sequencing (at baseline and at the end of the 12-month intervention) and fit regression (logistic and generalized linear) models to identify miRNAs related to metformin and insulin response. The overarching hypothesis of this application is that circulating miRNA profiles (reflecting tissue-specific miRNA changes) contribute to progression of beta-cell failure and insulin resistance during treatment with metformin and/or insulin in youth with dysglycemia. We will test the following aims: (1) To identify baseline circulating miRNAs associated with improvement or worsening in beta-cell function (insulin sensitivity- adjusted steady-state C-peptide and acute C-peptide response to arginine at maximal glycemic potentiation [ACPRmax]) and insulin sensitivity (mean glucose infusion rate divided by mean steady-state plasma insulin concentration [M/I]) at 12 months in youth with prediabetes or early T2D after a 12-month intervention with glargine + metformin or metformin alone (n=32) and (2) to identify changes in circulating miRNAs (between baseline and month 12) associated with improvement or worsening in beta-cell function and insulin sensitivity at 12 months in youth with prediabetes or early T2D after a 12-month intervention with glargine + metformin or metformin alone (n=32). Successful completion of the proposed work will identify circulating miRNAs related to progression of beta- cell failure and insulin resistance in youth. In addition, it will provide necessary pilot data that will inform design of larger studies to determine the epigenetic mechanisms contributing to progression of beta-cell failure and insulin resistance despite treatment with insulin and/or metformin in youth with prediabetes and early T2D. Identifying epigenetic mechanisms of metformin and insulin failure is a critical next step in the development of novel treatments to prevent pediatric T2D or induce remission of prediabetes.